EP0277697A1 - Antigène de polymyxine lipopolysaccharide et méthode associée - Google Patents

Antigène de polymyxine lipopolysaccharide et méthode associée Download PDF

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Publication number
EP0277697A1
EP0277697A1 EP88300051A EP88300051A EP0277697A1 EP 0277697 A1 EP0277697 A1 EP 0277697A1 EP 88300051 A EP88300051 A EP 88300051A EP 88300051 A EP88300051 A EP 88300051A EP 0277697 A1 EP0277697 A1 EP 0277697A1
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Prior art keywords
endotoxin
lipopolysaccharide
antigen
antibody
matrix
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German (de)
English (en)
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Meryl H. Karol
Lisa K. Ryan
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University of Pittsburgh
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University of Pittsburgh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia

Definitions

  • the present invention relates to a polymyxin lipo­polysaccharide antigen that creates antibodies that detect endotoxin and the method of making the antigen.
  • This invention relates to the production and characterisation of antigens that create antibodies specific for endotoxin and more specifically, the antigen and the antibodies, and use of these specific antibodies as an immunodiagnostic reagent to detect endotoxin.
  • Endotoxin Bacterial contamination is currently one of the leading causes of fatal bacterial infection. Endotoxin is known to cause toxic effects in multiple cell types and organs. Endotoxin is responsible for the symptoms assoc­iated with byssinosis, septicemia, bacteremia, and the like. These symptoms may include fever, diarrhea, hemor­rhagic shock, tissue damage, bronchospasms, damage to endothelial cells and the like. Release of endotoxin into the blood stream may cause endotoxin shock as well as other systemic effects.
  • Endotoxin is a component of the cell wall of Gram negaitve bacteria.
  • the cell wall of Gram negative bacteria is multilayered and quite complex.
  • Endotoxin is composed of lipid A and a polysaccharide.
  • Lipid A is the core materi­al.
  • the polysaccharide is exterior of the lipid A.
  • the polysaccharide component is unique to each type of bac­terium.
  • the LAL assay measures endotoxin by detecting only one biological activity of endotoxin in a highly purified sample.
  • the LAL measures the ability of lipopolysaccharide to gel or precipitate the lipote. Results from the assay do not correlate with the ability of endotoxin to cause respiratory toxicity in animals or neutrophil chemotaxis in human beings. Difficulties encountered with the LAL assay include interference by numerous organic compounds yielding both false-positive and false-negative results, as well as an inability to correlate consistently with the USP rabbit pyrogen test.
  • the rabbit pyrogen test involves injecting a rabbit with a sample suspected of endotoxin toxicity. The temperature of the rabbits is measured for a period of time. If a fever is produced in the rabbit, the presence of endotoxin is indicated. A gross estimate of the amount of endotoxin can be determined by the rise in temperature of the rabbit.
  • the USP rabbit test for pyrogenicity is expen­sive and the results are not always accurate.
  • Polyclonal antibodies have been produced to wild­type LPS by immunizing rabbits intravenously with heat­killed bacteria. See Carlsson et al, "Titration of Antibodies to Salmonella O Antigens by Enzyme-Linked Immunosorbent Assay” Infection and Immunity , Volume 6 (1972) pp. 703-708; Holmgren “Studies of Methods for Quantitation of Agglutinens and Precipitins of Escherichia Coli O and K Antigen” Int. Arch. Allergy, Volume 37 (1970) pp. 480-494. Additionally, covalent linking of the disaccharide antigenic determinants to bovine serum albumin (BSA) has also resulted in high titered antisera.
  • BSA bovine serum albumin
  • the present invention provides antigens that produce antibodies that are specific for the lipopolysaccha­ride in endotoxin.
  • a method of producing the antigen and the antibodies is also disclosed. Further, immunodiagnostic assays using these antibodies are also disclosed.
  • the antigen is produced by a polymyxin B-matrix complex (PMC) which is mixed with and which binds to free lipopolysaccharide.
  • PMC polymyxin B-matrix complex
  • the polymyxin B-matrix-LPS complex is separated from uncomplexed materials by centrifugation and then washed.
  • LPS polymyxin-B matrix antigen for immunization of animals yields a high titer of antibody.
  • the antibodies of the present invention are highly specific for the LPS portion of the complex and do not react with the polymyxin B component. This procedure produces antisera which detects and quantifies the amount of endotoxin in biological fluids.
  • Hybridomas secreting monoclonal antibodies specific for endotoxin can be produced by fusion of myeloma cells with spleen cells from mice immunized with purified endotoxin. Hybridomas secreting antibodies specific for endotoxin were selected and cloned. These hybridomas were then grown as ascitic tumors in mice and monoclonal anti­bodies were purified from the ascites fluids. Alternative­ly, the hybridomas may be grown in mass culture.
  • the present invention discloses an antigen which produces antibodies that are specific for lipopolysaccharide or endotoxin; a method for producing antigen that induces antibodies that are specific for the lipopolysaccharide or endotoxin and diagnostic tests using these antibodies is also disclosed.
  • a method of producing the antigen involves mixing a specific free LPS, such as a Gram-negative bacterial endo­toxin (LPS), with polymyxin B immobilized on a matrix for about 5 minutes to 24 hours at about 4°C to 37°C.
  • LPS-­polymyxin B-matrix complex is centrifuged to separate the complex from the supernatant which contains free LPS and then washed preferably with saline.
  • the complex is emulsi­fied with an immunostimulatory agent, such as Freund's Com­plete Adjuvant, and injected into an animal, such as guinea pigs, mice, rats, and the like.
  • the immunogen stimulates the production of high titer antisera specific for the LPS moiety.
  • Antibodies were highly specific for the immunizing LPS and showed no cross-reactivity with heterologous endotoxin.
  • the high titer of antibody produced following a single injection indicates that the method pro­duces a strong immunogenic stimulus such as that required for production of monoclonal antibodies.
  • Polymyxin B is a peptide antibiotic which contains a cationic cyclopeptide ring with a 7 or 8 carbon fatty acid attached through an amide bond. Polymyxin B has been shown to bind electrostatically and possible hydrophobically to LPS. This property has been utilized to neutralize the biological effects of endotoxin in vivo and to render solutions endotoxin-free. Polymyxin B has a high affinity for endotoxin.
  • the matrix be insoluble in an aqueous medium and be capable of binding to or reacting with Polymyxin B.
  • the matrix may be composed of synthetic, natural, or polymeric materials, such as resinous materials, such as plastics, for example, polyethylene, polypropylene, polystyrene and the like, cellulose, nitrocellulose, dextran, fibrous materials, such as graphite, asbestos, nylon and the like, for example.
  • the most preferred matrix is agarose.
  • Agarose is a natural seaweed product. It is in the form of an insol­uble gel. Agarose swells in water. Agarose has chemical groups on its surface to which proteins can be attached.
  • a preferred method of making the polymyxin B-­agarose complex uses agarose beads.
  • Agarose beads such as Affi-Gel 10 are an agarose derivative with added aliphatic arms about 10A in length, terminated with an active carboxy-N-hydroxysuccinimide ester.
  • the polymyxin B is covalently attached to the arms and suspended in a pre­servative, such as 50% glycerol and 0.5% sodium azide.
  • Free lipopolysaccharide is a bacterial extract which may be obtained from the bacterial wall using solvents such as phenol, chloroform, petroleum ether, butanol, tri­chloroacetic acid and the like, and purified.
  • the polysac­charide component in the bacterial cell wall is unique to each type of bacteria.
  • microorganisms that contain endotoxin include Pseudomonodaceae, Azotobactera­ceae, Rhizobiaceae, Methylomonadaceae, Halobacteriaceae, Acetobacteria, Enterobacteriaceae, Bibrionaceae, Bacteroida­ceae, Neisseriaceae, Veillonellaceae, Nitrobacteraceae, Siderocassaceae, and the like. More specifically, LPS may be obtained from Escherichia coli , Salmonella minnesota , Pseudomonas syringae , Enterobacter agglomerans , Klebsiella pneumonia , and the like.
  • Polymyxin B binds LPS very well by electrostatic interaction with possibly some contribution from hydrophobic interaction.
  • the matrix which is bonded to polymixin B, helps to purify the antigen. Due to the insolubility of the matrix, it functions to pull the LPS bound to polymyxin out of solution.
  • the preferred method of producing a hybridoma antibody is best illustrated by reference to Figure 1.
  • An animal is injected with LPS-PMC 2.
  • the spleen of the animal is removed.
  • myeloma cells 4 are cultured.
  • the spleen cells 7 and the myeloma cells 6 are fused in the presence of a fusion promoter, preferably in polyethylene glycol 8 for about 2 hrs. at 37°C.
  • the fused cells are then placed in a medium that will not support unfused cells 10, preferably HAT medium, for selection.
  • Cells that have successfully fused, also known as hybrids are selected by the HAT medium.
  • the hybrid cells are assayed for the presence of antibody secretion 12.
  • the preferred assay is an ELISA assay.
  • each positive culture may be frozen 14. Positive cultures are then cloned 16. Cloning may be done either by limiting dilution or by placing the positive cultures into mice and grown. The cells are then cultured in HAT medium and selected again 18. The positive cultures are then assayed for the presence of the antibody 20. The preferred assay is the ELISA assay.
  • sections of the positive cultures may be frozen 22. The positive cultures are recloned 24 and analyzed to select variants. The clones 28 finally selected are grown. Alternatively, they may be frozen and thawed. At this point the hybridomas may be grown in mass culture 32, thereby producing the desired monoclonal antibody 34.
  • an animal such as a guinea pig, mouse, or rat may be injected with the hybridoma 36 to induce myelomas that secrete the antibody 38.
  • the above-described antigen and antibodies may be used in a diagnostic test to determine the presence of endo­toxin in a specimen, for example, human secretions, urine, food, blood, serum, and the like. Due to the specificity of the antibodies, the samples do not have to be purified when using the diagnostic tests.
  • the antigen and antibodies may be used to detect the presence of endotoxin in pharmaceuti­cals of any type and medical devices, such as hemodialysis membranes, plasma fractionation devices, and catheters and the like.
  • the antibody may be immobilized on a filter to determine the presence of endotoxin in the air.
  • the antibody may be incorporated into an immuno­diagnostic test, such as ELISA, RIA and the like, and the endotoxin detected by competitive inhibition assay.
  • an immuno­diagnostic test such as ELISA, RIA and the like
  • samples of suspected endotoxin material are placed in at least some of the wells of assay plates, previously coated with endotoxin, and incubated, then washed with buffer.
  • Antibody is added to the wells and allowed to react.
  • the wells are washed again.
  • An anti-immunoglobulin-­enzyme is added to the wells and allowed to react with the monoclonal antibody.
  • the enzyme portion may be, for example, horseradish peroxidase, or alkaline phosphatase. The wells are washed.
  • a chromogenic substrate such as p -­nitrophenyl phosphate, o -dianisidine with hydrogen peroxide, 5-aminosulfacylic acid with hydrogen peroxide, 2,2-azino-­di(3-ethyl benzthiazoline sulfonic acid) (ABTS) with hydro­gen peroxide for example, is added to each well and then allowed to react. The reaction is stopped, the color of the chromogen may be measured visually or spectrophotometrically to determine the presence and quantity of endotoxin.
  • ABTS 2,2-azino-­di(3-ethyl benzthiazoline sulfonic acid)
  • a plate having 96 wells may be coated with antibody incubated and washed. Excess binding sites on the plates may be blocked with a blocking solution containing heterologous proteins such as bovine serum albumin or milk proteins. The blocing solution is removed and the well is washed. Samples containing the suspected endotoxin are added to the wells and incubated. The plates are washed. The antibody to which an enzyme such as horse­radish peroxidase or alkaline phosphatase has been attached is added to the wells and reacts with the bound endotoxin. The wells are washed and conjugate is added.
  • the chromo­ genic substrate such as 0.1% p-nitrophenyl phosphate, 10% diethanolamine, 0.01% MgCl2, pH 9.6 in the example of alkaline phosphatase is added and the reaction is stopped with any strong basic compound which raises the pH high enough to inactivate the enzyme by denaturation. Results may be read visually or spectrophotometrically to show the presence and quantity of endotoxin.
  • a competitive ELISA procedure may also be used. This procedure is illustrated in Figures 2a and 2b.
  • a solution of suspected endotoxin 40 or LPS in 0.02 M phosphate buffer is placed in a test tube with antibody 42.
  • This test solution 44 is then added to wells precoated with LPS 46. If endotoxin is present in test solution 44, the antibody will bind to the endotoxin 47.
  • the antibody 42 also binds to the LPS coated on the plate 46, but when endotoxin is present in the solution, there is competition for the antibody 42.
  • the plate 42 is washed with phosphate buffer 48 removing the bound endotoxin and antibody 47 present.
  • An enzyme substrate which is hydrolyzed by the enzyme is then added to the wells 50. The amount of color produced by addition of a chromogenic substrate which binds to the enzyme indicates the presence and quantity of endo­toxin.
  • Endotoxin or LPS may be adsorbed to a solid medium 70, such as a paper disk and the above procedure followed.
  • a solid medium 70 such as a paper disk and the above procedure followed.
  • endotoxin is present in the test solution 74 which contains antibody 72.
  • the antibody binds to the endotoxin in solution 78.
  • the antibody also binds to the bound LPS (not shown), but the bound LPS is competing with the endotoxin in the test solution for the antibody.
  • the plate is washed 80, thereby washing away the bound antibody and endotoxin in the test solution.
  • I125-antibody 82 is then added and allowed to react with the bound antibody.
  • the antibody 72 binds to the LPS bound to the plate 88.
  • the labeled antibody 90 binds to the antibody complex 92.
  • the amount of radioactivity bound to each solid medium may be measured using a gamma scintillation counter. In the case of endotoxin presence, the amount of I125-antibody bound to the plates is inhibited. If no endotoxin is present in the test solution as shown in Figure 3b, the level of radioactivity is higher.
  • a known quanti­ty of I125-antibody can be added so a LPS-plate in the presence of an unknown amount of test antibody.
  • the ability of the test antibody to complete with the I125-antibody indicates the amount of antibody in the test solution.
  • E. Coli 055:B5 LPS (Difco Laboratories, Detroit, MI) was coupled to polymyxin B-agarose (Sigma Chemical Company, St. Louis, MO) using the following procedure. A 10 ml polymyxin B-agarose suspension of beads was washed to rid the polymyxin B-agarose beads of the glycerol and sodium azide preservative, resulting in about 5 ml of polymyxin B and agarose. One ml of a 10 mg/ml solution of E. coli 055:B5 LPS in pyrogen-free sterile water (Abbott Labora­tories, North Chicago, IL) was mixed with the polymyxin B-­agarose beads.
  • pyrogen-free sterile water Abbott Labora­tories, North Chicago, IL
  • the LPS was added in greater than 10 fold excess. The mixture was rocked gently overnight at 4°C. The LPS polymyxin B-agarose complex was centrifuged at 470 x g for 10 min., then washed twice with 0.9% saline. For immunization, the gel was mixed with a small amount of saline and then emulsified with Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI).
  • Serum samples were screened for antibody to E. coli 055:B5 LPS using an immunobinding assay.
  • LPS from Escherichia coli 055:B5 was adsorbed onto nitrocellulose paper discs (0.45 ⁇ m Bio-Rad Laboratories, Richmond, CA) using the following adsorptive immobilization technique.
  • Test serum (diluted 1:5) was added to each disc. After shaking the discs for about 2 hrs. at room temperature they were washed four times. Rabbit anti-guinea pig IgG (H + L)-horseradish peroxidase conjugate (Miles Scientific, Naperville, IL) (1:100) was then added and incubation con­tinued for about 2 hrs. at room temperature. The discs were washed three times. One ml of 4-chloro-1-naphthol hydrogen peroxide substrate solution was then added. The discs were incubated for 5 mins. Following visualization of color by eye, the substrate solution was removed and the discs stored at 4°C in distilled water. Reaction was visualized for at least 3 months.
  • Figure 4 shows dot immunoblotting of sera from E. coli 055:B5 LPS-PAC immunized guinea pigs. Rows A and B had preimmunized sera. Rows C and D had immune sera from day 29. Figure 4 indicates a positive reaction on nitrocellu­lose discs containing E. coli 055:B5 LPS antigen and immune serum from each of the 5 guinea pigs (row D). No staining was obtained with any of the pre-immunization sera (rows A and B) or with discs containing the polymyxin B carrier (row C).
  • Antibody titers were determined using the fol­lowing ELISA procedure.
  • Wells of Nunc Immunoplate I high binding capacity microtitration plates, Nunc Intermed, Inc., Kamstrup, Denmark
  • Plates were washed 3 times with saline tween (0.85% NaCl and 0.05% Tween 20).
  • Serum in PBS-Tween containing 0.5% BSA was added to the plates. The plates were incubated for 2 hrs. at 37°C.
  • IgG antibody was determined using rabbit anti-guinea pig IgG (H + L) alkaline phosphatase conjugate (Miles Scientific, Naperville, IL). After incuba­tion at room temperature for 16 hrs., the enzyme was assayed using disodium p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, MO) in 1 M diethanolamine and 0.5 mM MgCl2. Following reaction for 30 mins. at 37°C, 100 ⁇ l of 1 M NaOH were added and the optical density was read at 410 nm using an MR 600 Microplate reader (Dynatech Laboratories, Inc., Alexandria, VA).
  • the titer of antibody in each serum was determined using ELISA. Microtiter plates coated with 0.001-50 ⁇ g/ml polymyxin B showed no titer in 4 of 5 animals. One animal had a titer of 1:10 only when 50 ⁇ g/ml of antigen was used to coat the plates.
  • microtiter plates were coated with 0.01 to 10 ⁇ g/ml LPS.
  • the optimal concentration of LPS to detect antibody was determined to be 10 ⁇ g/ml.
  • the antibody content of serial serum dilutions for one animal following a booster immuni­zation is shown in Figure 5.
  • the titer of this serum was 1:3200.
  • Each of the other four guinea pigs gave similar absorbance curves.
  • Figure 6 shows blood obtained before booster immunization (Day 29) and following the booster immunization (Day 47).
  • the highest titer in the three animals was 1:6400.
  • the booster immunization in­creased this titer in two of these animals to 1:25,000 and also increased the LPS-specified antibody titer in another animal from 1:400 to 1:3200. In the two remaining animals, the booster immunization had little or no effect.
  • the specificity of the antibody was evaluated using an ELISA inhibition assay.
  • Sources of inhibitors were E. coli 055:B5( obtained from Difco Laboratories,Detroit, M1) E. coli 0111:B4, E. coli J5, Salmonella abortus equi , and Salmonella minnesota Re595 (all obtained from Sigma Chemical Company, St. Louis, MO).
  • the assay was similar to the ELISA method described hereinbefore.
  • the dilution of antiserum used was that which gave an absorbance of 0.25 in the ELISA.
  • the amount of inhibitor added to each well was varied. This amount ranged from 0.01 ⁇ g to 50 ⁇ g per well. Assays were performed in triplicate.
  • ELISA inhibition was used to evaluate the specifi­city of the antibodies and to determine whether antibodies were directed toward the lipid A region of the LPS molecule or to the sugar moieties.
  • Figure 7 illustrates a typical ELISA inhibition curve. Serum from guinea pig 4 obtained on day 47 showed specificity for the homologous E. coli 055:B5 LPS. The amount of inhibition increased with increased amount of E. coli 055:B5 LPS inhibitor from 0.01 ⁇ g to 5 ⁇ g. No inhibition was observed when LPS from E. coli 0111:B4, E. coli J5, Salmonella minnesota Re595, or Salmonella abortus equi were used in the system, indicating specificity for the E. coli 055:B5 LPS.
  • Table 1 summarizes the amount of inhibitor required to produce 50% inhibition in three antisera ob­tained before the booster immunization. Inhibition was obtained using the 70-495 ng E. coli 055:B5. By contrast, up to 50,000 ng of heterologous LPS could not produce any inhibi­ tion. These results indicated a high specificity of the antisera for the homologous LPS.
  • E. coli 055:B5 LPS was diluted with 0.1 M carbonate buffer, pH 9.6 to a concentration of 10 ⁇ g/ml. 100 ⁇ l of the LPS solution was added to each well of a 96 well poly­styrene plate. The plate was incubated for 3 hours at 37°C, then incubated over night at 4°C. The plate was washed three times for three minutes with Tween-Saline.
  • Antisera were diluted 1:100 in PBS-Tween BSA buffer, pH 7.4, and two fold dilutions were made in PBS-­Tween-BSA buffer up to 204,800. A test solution containing endotoxin was added. The diluted antisera and test solution were incubated in the wells for 2 hours at 37°C. The plates were then washed three times for three minutes each with Tween-Saline.
  • Rabbit anti-guinea pig IgG (H+L)-alkaline phosphatase enzyme conjugate was diluted 1:800 in PBS-­Tween. 100 ⁇ l was placed in each well and incubated overnight at room temperature. The plates were washed three times for three minutes each with Tween-Saline. Color was developed adding 100 ⁇ l of a 1 mg/ml substrate solution of p -nitrophenyl phosphate in 1M diethanolamine and 0.5 mM MgCl2 and incubated in each well for 30 minutes at 37°C. The reaction was stopped by addition of 100 ⁇ l per well of 1 N sodium hydroxide. The results were determined using an ELISA spectrophotometer at 410 nanometers.
  • the LPS-polymyxin B-agarose antigen produced antibodies specific for the LPS from E. coli 055:B5. While E. coli 055:B5 LPS has been used as an example, the method of this invention may be used to produce antigens and antibodies for other microorganisms containing endotoxin. In addition, the method of producing the antigen results in a purified product in an efficient, cost effective manner. Further, the diagnostic tests using the LPS and/or antibodies specific for the endotoxin allows assay of complex biological materials without any purifica­tion and no interference from foreign materials, with highly sensitive results.

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EP88300051A 1987-01-05 1988-01-05 Antigène de polymyxine lipopolysaccharide et méthode associée Withdrawn EP0277697A1 (fr)

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WO1995031730A1 (fr) * 1994-05-18 1995-11-23 Microquest Diagnostics, Inc. Procede de detection rapide d'un analyte
GB2325233A (en) * 1997-05-16 1998-11-18 Norsk Hydro As Substrates having bound polysaccharides and bacterial nucleic acids
WO2000017653A2 (fr) * 1998-09-17 2000-03-30 Labor Diagnostik Gmbh Leipzig Composition d'antigenes contre les salmonelles et kit permettant de detecter des anticorps contre des salmonelles
US6087342A (en) * 1998-05-15 2000-07-11 Fmc Biopolymer As Substrates having bound polysaccharides and bacterial nucleic acids
WO2002076343A1 (fr) * 2001-03-26 2002-10-03 Applied Research Systems Ars Holding N.V Methode et trousse pour quantification de polypeptides

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JPH04270965A (ja) * 1990-05-18 1992-09-28 Burton W Blais オリゴペプタイド吸着担体の調製方法、及びこれを使用したリポ多糖類の検定と除去方法
EP0472467A3 (en) * 1990-08-20 1993-03-17 Chiba Flour Milling Co. Ltd. Lps-containing analgesics and veterinary analgesics
US5496700A (en) * 1993-08-06 1996-03-05 The United States Of America As Represented By The Secretary Of The Navy Optical immunoassay for microbial analytes using non-specific dyes
IES65976B2 (en) * 1994-03-01 1995-11-29 Teagasc Agric Food Dev Authori Rapid detection of bacteria in liquid cultures
US5807694A (en) * 1995-09-07 1998-09-15 Economic Innovation And Technology Council, University Of Manitoba Detection of salmonella enteritidis and other pathogenic microorganisms and monoclonal antibody useful therefor
DE60326988D1 (de) * 2002-12-20 2009-05-14 Charlotte Mecklenburg Hospital Wegwerf-handvorrichtung zum auffangen von ausgeatmetem atemkondensat
US7828741B2 (en) * 2002-12-20 2010-11-09 The Charlotte-Mecklenburg Hospital Authority Utilizing lipopolysaccharide in exhaled breath condensate to diagnose gram negative pneumonia
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